Neuroprotective Effects Of Raloxifene On Aβ_25-35-induced Damages Via Mapk Signaling Pathway

Objective:Deficiency of ovarian hormones such as estrogen during the menopausal transition and early postmenopause is associated with a greater risk of memory decline, including mild cognitive impairment and AD. The neurotoxicity of Aβhas been implicated as a critical cause in the pathogenesis of AD. RLX, a kind of selective estrogen receptor modulators, might possess the neuroprotective role similar to estrogen in some studies. But there is little understanding about the mechanism of this protection. In this study, we evaluated the neuroprotective effects of RLX on Aβ_25-35-induced toxicity and investigated its potential mechanisms in cultured rat hippocampus neurons and PC12 cells.Methods:The cultured hippocampus neurons were divided into 5 groups: control group, Aβgroup, estrogen intervention group, benzogynestry intervention group, RLX intervention group. The cell viability was evaluated by MTT assay and the cell apoptosis was analyzed by flow cytometry with Annexin V/FITC-PI staining after 24h cultured with different drugs.The cell viability of PC12 cells treated by Aβ_25-35 (30μmol/L ) and/or RLX(10-6mol/L) was evaluated by MTT assay. The variation of cell death(necrosis and apoptosis) were analyzed using flow cytometry with Annexin V/FITC-PI staining. TheΔψm of the inner mitochondrial membrane was measured by fluorescence microscopy and flow cytometry with JC-1 staining. After treated with PD98059 (inhibitor of ERK1/2), SB230580 (inhibitor of p38MAPK) or SP600125 (inhibitor of JNK), the activation of ERK1/2, p38MAPK, JNK and caspase-9 were determined by Western blotting.Resultes:Compared to control group, the cell viability for Aβgroup decreased significantly (P<0.01), and the cell apoptosis for Aβgroup increased significantly (P<0.01). For estrogen intervention group, benzogynestry intervention group and RLX intervention group, MTT reduction increased significantly compared to Aβgroup (P<0.01,P<0.05,P<0.05), and the number of apoptotic cells decreased significantly compared to Aβgroup (P<0.05).RLX, in combination with Aβ_25-35 increased the cell viability (P<0.001), and reduced the number of apoptotic cells (P<0.05). RLX attenuated Aβ_25-35-induced the loss ofΔψm P<0.01). The changing ofΔψm was similar to the variation of apoptosis. PD98059 inhibited the effecs of RLX on cell viability and phosphorylation of caspase-9, an important molecule in the mitochondria pathway. No significant difference of cell viability or phosphorylation of caspase-9 had been found when PC12 cells were incubated with SB203580 and SP600125. Aβ_25-35 induced a time-dependent phosphorylation of p38MAPK and JNK. Treated the PC12 cells with RLX solitary, ERK1/2 was activated (P<0.01). Treated the PC12 cells with Aβ_25-35 and RLX, Aβ_25-35-induced phosphorylation of p38MAPK and JNK were inhibited (P<0.01 and P<0.001, respectively).Conclusions:RLX, as same as estrogen, attenuates Aβtoxicity on cultured rat hippocampus neurons and PC12 cells. RLX inhibited Aβ_25-35-induced PC12 cells apoptosis by activating of ERK1/2 pathway. RLX also attenuated Aβ_25-35-induced activation of p38MAPK and JNK. The mitochondria pathway was involved in this inhibitory effect by RLX.